(Fred)erick's Matsen's notes

• Coxsackie_B_virus
• Vesicular_stomatitis_virus

They are interested in:

• Identification of novel regulators of enterovirus infection by RNAi screening
• identification of novel regulators of panviral infection
• Novel components in the innate immune signaling (Actin associated component)
• Enteroviruses: how do they mediate immune signaling?

Cellular barriers to virus entry:

• Blood-brain barriers
• Airway epithelium
• GI epithelium

What about the placenta? Very critical barrier– stops trans-generational transmission.

Placenta formed from specialized epithelial cells. sCTB– multi-nucleated, contacts maternal blood. SYN– next to baby.

Going to focus on sCTBs. Not present in tissue culture collections. Model cell type: PHTs isolated from normal term deliveries.

PHTs are resistant to virus infection. Hard to find viruses that will replicate to any appreciable degree. Not just because of inefficient viral binding or entry.

Do PHT cells secrete an antivrial factor? Culture PHT cells and collect conditioned medium. Try to infect permissive cells on those media.

This conditioned medium protects lots of different types of cells. Specific to PHT cells. Not due to direct neutralization. Correlates to significant reductions in viral titers. Works for lots of cell types.

They call it “factor Y” and try to figure out what’s going on. Not a type I interferon. Can completely remove IFN pathway. Not NF-kB. Not cell death.

Sonication removes the antiviral effects of conditioned PHT medium (!!). So exosomes:

• Sensitive to sonication
• Formed inside the cell in multivesicular endosomes
• Contain protein and RNA molecules
• miRNA packages in exosomes can impact recipient
• PHT cells release very high levels of exosomes.

Purify exosomes, and they can suppress viral infection.

Is there something special about PHT exosomes? Yes. They are the only ones they have seen to be antiviral.

What is in the exosomes? Placenta epxresses high levles of a primate-specific miRNA cluster: C19MC. Function unknown. Found within a week of conception. Disappear 24hrs post delivery. Can be found in fetal blood too.

Pofiling miRNAs. Linear relationship between expression in exosomes and expression in PHT cells. They are only expressed in PHT cells. They do block infection.

How are these miRNA delivered. RNAseq shows that they appear in recipient cells.

46 members of this miRNA family. Order mimics up and do transfections. The ones that are most abundant are also the most protective. Focus on top 7– just these are very protective.

Does it work by autophagy? These effects are not virus-specific. Makes 2x membrane vesicles called autophagosomes -> autolysosomes.

Indeed it does. Can see using fluorescence and EM.

The miRNAs induce autophagy. Specifically, result of autophagic induction. Also, inhibiting autophagy suppesses the antiviral effects of the miRNAs. More generally, can enhance viral replication by inhibiting autophagy.

How could autophagy limit infection? PHTs make exosomes that get taken into cells. Robust association between virus and autolysosomes.

“TORCH” viruses hurt fetuses. Toxoplasma gondii, Rubilla, CMV, HSV-2. CMV infection robustly enhanced by their miRNAs! All other viruses down. Toxoplasma gondii also enhanced by miRNA expression.

Now they have mouse model. What are targets?

• Q: Is the function of these miRNAs to be antiviral.

• A: She doesn’t think so. Babies do lots of autophagy just to feed themselves.

• Q: What about stimulating autophagy by other means.

• A: Depends, and it seems to be dependent on the means of inducing autophagy.

• Q: When does this effect start in pregnancy?

• A: Don’t know. Tough to get early placentas.

• Q: miRNAs in other species?

• A: These miRNAs are only in primates.

• Q: Other factors in exosomes?
• A: Probably.